Normalization of polymerase activity

ABSTRACT

Provided herein is technology relating to the amplification-based detection of nucleic acids and particularly, but not exclusively, to methods and compositions for minimizing variability in the activity between different samples or manufacturing lots of DNA polymerases, such as Taq DNA polymerase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation U.S. patent application Ser.No. 14/036,649 filed Sep. 25, 2013 claims the benefit of U.S.Provisional Patent Application 61/705,603, filed Sep. 25, 2012, each ofwhich is incorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided herein is technology relating to the amplification-baseddetection of nucleic acids and particularly, but not exclusively, tomethods and compositions for altering the behavior of calibrationstandards to mimic natural samples and minimize variability in theactivity of manufactured polymerases such as Taq DNA polymerase.

BACKGROUND

Methods for the quantification of nucleic acids are important in manyareas of molecular biology and in particular for molecular diagnostics.At the DNA level such methods are used, for example, to determine thecopy numbers of gene sequences amplified in the genome. Further, methodsfor the quantification of nucleic acids are used to determine mRNAquantities as a measure of gene expression.

Among the number of different analytical methods that detect andquantify nucleic acids or nucleic acid sequences, variants of thepolymerase chain reaction (PCR) have become the most powerful andwidespread technology, the principles of which are disclosed in U.S.Pat. Nos. 4,683,195; 4,683,202; and 4,965,188. Automated methods ofamplification typically rely on the use of thermostable DNA polymerases,e.g., from Thermus aquaticus and other thermophilic organisms, orengineered by truncation, modification, or chimerization of proteinsfrom thermophilic organisms. Accordingly, many manufacturers now producethermostable polymerases from a variety of sources. For example, the DNApolymerase I from Thermus aquaticus (“Taq” polymerase) is produced by anumber of different manufacturers and one concern is the variability inthe activity of such enzymes between the producers of the enzyme, and/orbetween different lots of enzyme from a single producer.

In order to minimize the consequences of spurious reactions attemperatures below the optimal PCR conditions, such as during reactionset up, a number of methods have been developed for suppressing theactivity of the Taq polymerase prior to the desired start of thereaction, known collectively as “hot start PCR” or “hot start Taq.”These include chemical modification of the Taq (U.S. Pat. Nos. 5,677,152and 5,773,258), binding antibody to the Taq enzyme (U.S. Pat. Nos.5,338,671 and 5,587,287), and aptamer binding to the Taq (U.S. Pat. Nos.5,693,502 and 6,020,130, as well as others methods of hot start. One ofthe practical consequences of hot start technology is that assaying andadjusting the activity of the Taq enzyme becomes technically morechallenging for Taq enzyme vendors.

In addition, the maturation of PCR and related amplification techniqueshas produced powerful technologies to detect nucleic acids withincreasing accuracy and precision. These techniques require thatvariability in the activity of polymerase be minimal, not only as itvaries between suppliers but also as it varies among lots of a singlesupplier's production runs. As such, minimizing the variability ofpolymerase activity in nucleic acid detection reactions is a problem forsome amplification methods.

SUMMARY

In the course of development of methods described herein, it has beendetermined that different preparations of enzymes, e.g., differentmanufacturing lots of Taq polymerase from the same vendor, have produceddifferent results as judged by variation in the standard curvesgenerated for quantitative analysis. For example, during the developmentof a quantitative allele-specific real-time target and signalamplification (QuARTS) assay (see, e.g., U.S. Pat. Appl. Pub. No.2009/0253142 or U.S. Pat. No. 8,361,720), using combined targetamplification and FEN-1-mediated signal amplification, and related todetecting nucleic acids associated with colon cancer, such variation wasmanifested as an observed difference in the slopes and/or intercepts ofthe standard curves that changed as a function of Taq polymerasemanufacturing lot. In these types of assays, standard curves are used toquantify the number of target DNAs in the assay sample; thus, variationsin the standard curves may produce errors in the copy number calculatedfor the particular DNA that is evaluated by the test. Surprisingly, thelot-to lot variability of the polymerase enzyme could not be compensatedfor by adjusting the amounts of polymerase added to the reactions.

Accordingly, provided herein is technology related to a method forminimizing enzyme variability in an assay sample or reaction mixturecomprising a target nucleic acid a DNA polymerase, a flap endonuclease,and a hairpin oligonucleotide, the method comprising adding purified,exogenous, non-target nucleic acid, e.g., DNA, to the reaction mixture;and measuring the amount of the target nucleic acid in the reactionmixture. In preferred embodiments, the reaction mixture furthercomprises a primer. In certain particularly preferred embodiments, thehairpin oligonucleotide comprises a FRET cassette oligonucleotide. Incertain embodiments, the target nucleic acid is human. In preferredembodiments, the target nucleic acid is DNA.

In some embodiments, purified non-target DNA included in the assayreaction mixture is isolated from fish, e.g., herring, cod, and/orsalmon. In some preferred embodiments, the non-target DNA is a mixtureof DNA isolated from different sources. For example, in some embodimentsthe DNA is isolated from two or more different kinds of fish, e.g., amixture of fish such as herring, cod, and/or salmon. Embodiments alsoprovide non-target nucleic acid isolated from mammals, fish, birds,amphibians, etc. In some embodiments, the non-target DNA comprises DNAisolated from mouse.

In some embodiments, the non-target nucleic acid is added at aconcentration of approximately 2 to approximately 20 nanograms per μl ofthe reaction mixture. In certain embodiments, the non-target nucleicacid is added at a concentration of approximately 6 to 7 nanograms perμl of reaction mixture volume.

In some embodiments, the target nucleic acid is a calibrator or astandard, e.g., in a dilution series for quantitative amplificationreactions. In some embodiments, measuring the amount of the targetnucleic acid in the reaction mixture comprises use of a PCR-invasivecleavage assay, such as a QuARTS assay. In some embodiments, the targetnucleic acid is a human nucleic acid, e.g., in some embodiments, humanDNA. Furthermore, some embodiments provide that the target nucleic acidcomprises human genomic DNA.

In another aspect, the technology relates to a composition comprisinghuman genomic DNA; an oligonucleotide specific for the human genomicDNA; a DNA polymerase; a flap endonuclease; a FRET cassette, andpurified exogenous non-target DNA. In some embodiments the polymerase isTaq polymerase. In some embodiments, the extraneous, non-target DNA isisolated from fish.

In some embodiments of the technology, the DNA polymerase used comprisesa thermostable DNA polymerase. In certain embodiments, the thermostableDNA polymerase is a eubacterial DNA polymerase, and in some preferredembodiments, the eubacterial DNA polymerase is Taq DNA polymerase, i.e.,is isolated from Thermus aquaticus. In certain preferred embodiments,the DNA polymerase is modified for hot start PCR.

In some embodiments, the flap endonuclease used comprises a FEN-1endonuclease, and in certain embodiments, said FEN-1 endonuclease isfrom an archaeal organism. In particularly preferred embodiments, theflap endonuclease is thermostable.

The technology also provides reaction mixtures configured for minimizingenzyme variability in performance of an assay. In some embodiments,reaction mixtures of the technology comprise target nucleic acid;purified exogenous non-target DNA, and PCR-invasive cleavage assayreagents comprising, e.g., thermostable DNA polymerase, dNTPs; a firstprimer and a second primer configured for amplifying a product from thetarget nucleic acid; a flap endonuclease, a FRET cassette, and a flapoligonucleotide, wherein the reaction mixture is characterized in thatit can amplify the target nucleic acid and produce a detectable signalproportional to the amount of target nucleic acid in the mixture.

In some configurations, the technology provides a set of reactionmixtures, each of said reaction mixtures in the set comprisingPCR-invasive cleavage assay reagents comprising thermostable DNApolymerase; dNTPs; a first primer and a second primer configured foramplifying a product from said target nucleic acid; a flap endonuclease,a FRET cassette, and a flap oligonucleotide. In certain embodiments,each reaction mixture in the set of reaction mixtures is characterizedin that it can amplify the target nucleic acid and produce a detectablesignal proportional to the amount of said target nucleic acid in thatreaction mixture. In preferred embodiments, the set of reaction mixturescomprises a dilution series in which each member of the dilution seriescomprises a different known amount of the target nucleic acid, andessentially the same concentrations of the components of thePCR-invasive cleavage assay reagents.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 provides a schematic diagram of a combined PCR-invasive cleavageassay.

FIGS. 2A and 2B are plots showing that addition of herring DNA decreasesthe differences between the slopes and intercepts among samples analyzedwith two different lots of Taq polymerase in a QuARTS assay. Thereactions had 0, 1 or 4 ng of herring DNA per 30 μl reaction.

FIGS. 3A and 3B are plots comparing the effect of adding tRNA (FIG. 3A)to the effect of adding herring DNA (FIG. 3B) on the linear log fit ofdata from sets calibration reactions performed using two different lotsof Taq DNA polymerase.

FIGS. 4A-4F are a series of plots showing that the addition of 200 ng offish DNA per 30 μl reaction produced nearly identical plots using twodifferent lots of Taq DNA polymerase “DEV” and “Pnew”. The differentlots of Taq polymerase were tested at 0.05 u/μl of reaction volume (4A,4B and 4C) and 0.07 units/μl of reaction volume (4D, 4E, and 4F), inQuARTS assays as described below.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the technology may be readilycombined, without departing from the scope or spirit of the technology.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

As used herein in reference to non-target DNA, the term “exogenous”refers to non-target DNA that is isolated and purified from a sourceother than the source or sample containing the target DNA. For example,purified fish DNA is exogenous DNA with respect to a sample comprisinghuman target DNA. Exogenous DNA need not be from a different organismthan the target DNA. For example, purified fish DNA obtainedcommercially would be exogenous if added to a reaction configured todetect a target nucleic acid in a sample from a particular fish. Inpreferred embodiments, exogenous DNA is selected to be undetected by anassay configured to detect and/or quantify the target nucleic acid inthe reaction in to which the exogenous DNA is added.

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated. An oligonucleotide “primer” mayoccur naturally, as in a purified restriction digest or may be producedsynthetically. In some embodiments, an oligonucleotide primer is usedwith a template nucleic acid and extension of the primer is templatedependent, such that a complement of the template is formed.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S.Pat. No. 5,494,810; herein incorporated by reference in its entirety)are forms of amplification. Additional types of amplification include,but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No.5,639,611; herein incorporated by reference in its entirety), assemblyPCR (see, e.g., U.S. Pat. No. 5,965,408; herein incorporated byreference in its entirety), helicase-dependent amplification (see, e.g.,U.S. Pat. No. 7,662,594; herein incorporated by reference in itsentirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and5,338,671; each herein incorporated by reference in their entireties),intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al.,(1988) Nucleic Acids Res., 16:8186; herein incorporated by reference inits entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al.,Nucleic Acids Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169;each of which are herein incorporated by reference in their entireties),methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821-9826; herein incorporated by reference in its entirety), miniprimerPCR, multiplex ligation-dependent probe amplification (see, e.g.,Schouten, et al., (2002) Nucleic Acids Research 30(12): e57; hereinincorporated by reference in its entirety), multiplex PCR (see, e.g.,Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156;Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al.,(2008) BMC Genetics 9:80; each of which are herein incorporated byreference in their entireties), nested PCR, overlap-extension PCR (see,e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367;herein incorporated by reference in its entirety), real time PCR (see,e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al.,(1993) Biotechnology 11:1026-1030; each of which are herein incorporatedby reference in their entireties), reverse transcription PCR (see, e.g.,Bustin, S. A. (2000) J. Molecular Endocrinology 25:169-193; hereinincorporated by reference in its entirety), solid phase PCR, thermalasymmetric interlaced PCR, and Touchdown PCR (see, e.g., Don, et al.,Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485; eachof which are herein incorporated by reference in their entireties).Polynucleotide amplification also can be accomplished using digital PCR(see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004,(1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41,(1999); International Patent Publication No. WO05023091A2; US PatentApplication Publication No. 20070202525; each of which are incorporatedherein by reference in their entireties).

The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic or other DNA or RNA, withoutcloning or purification. This process for amplifying the target sequenceconsists of introducing a large excess of two oligonucleotide primers tothe DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (“PCR”). Because thedesired amplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified” and are “PCR products” or “amplicons.” Those of skillin the art will understand the term “PCR” encompasses many variants ofthe originally described method using, e.g., real time PCR, nested PCR,reverse transcription PCR (RT-PCR), single primer and arbitrarily primedPCR, etc.

As used herein, the term “nucleic acid detection assay” refers to anymethod of determining the nucleotide composition of a nucleic acid ofinterest. Nucleic acid detection assay include but are not limited to,DNA sequencing methods, probe hybridization methods, structure specificcleavage assays (e.g., the “INVADER” flap assay, or invasive cleavageassay, (Hologic, Inc.) described, e.g., in U.S. Pat. Nos. 5,846,717,5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev etal., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272(2000), and in combined PCR/invasive cleavage assays (Hologic, Inc.,e.g., in U.S. Patent Publications 2006/0147955 and 2009/0253142), eachof which is herein incorporated by reference in its entirety for allpurposes); enzyme mismatch cleavage methods (e.g., Variagenics, U.S.Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated byreference in their entireties); polymerase chain reaction (PCR),described above; branched hybridization methods (e.g., Chiron, U.S. Pat.Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporatedby reference in their entireties); rolling circle replication (e.g.,U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporatedby reference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818,herein incorporated by reference in its entirety); molecular beacontechnology (e.g., U.S. Pat. No. 6,150,097, herein incorporated byreference in its entirety); E-sensor technology (U.S. Pat. Nos.6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporated byreference in their entireties); cycling probe technology (e.g., U.S.Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated byreference in their entireties); Dade Behring signal amplificationmethods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230,5,882,867, and 5,792,614, herein incorporated by reference in theirentireties); ligase chain reaction (e.g., Barany Proc. Natl. Acad. SciUSA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S.Pat. No. 5,288,609, herein incorporated by reference in its entirety).

In some embodiments, target nucleic acid is amplified (e.g., by PCR) andamplified nucleic acid is detected simultaneously using an invasivecleavage assay. Assays configured for performing a detection assay(e.g., invasive cleavage assay) in combination with an amplificationassay are described in US Patent Publication 20090253142 A1(application. Ser. No. 12/404,240), incorporated herein by reference inits entirety for all purposes, and as diagrammed in FIG. 1. Because manycopies of the FRET cassette are cleaved for each copy of the targetamplicon produced, the assay is said to produce “signal amplification”in addition to target amplification. Additional amplification plusinvasive cleavage detection configurations, termed the QuARTS method,are described in U.S. Pat. No. 8,361,720, and in U.S. patent applicationSer. Nos. 12/946,745; and 12/946,752, incorporated herein by referencein their entireties for all purposes.

As used herein, the term “PCR-invasive cleavage assay” refers to anassay in which target nucleic acid is amplified and amplified nucleicacid is detected simultaneously using a signal-amplifying invasivecleavage assay employing a FRET cassette, and in which the assayreagents comprise a mixture containing DNA polymerase, FEN-1endonuclease, a primary probe comprising a portion complementary to atarget nucleic acid, and a hairpin FRET cassette. PCR-invasive cleavageassays include the QuARTS assays described in U.S. Pat. No. 8,361,720,and in U.S. patent application Ser. Nos. 12/946,745; and 12/946,752, andthe amplification assays of US Patent Publication 20090253142 A1 asdiagrammed in FIG. 1.

As used herein, the term “PCR-invasive cleavage assay reagents” refersto one or more reagents for detecting target sequences in a PCR-invasivecleavage assay, the reagents comprising nucleic acid molecules capableof participating in amplification of a target nucleic acid and information of an invasive cleavage structure in the presence of thetarget sequence, in a mixture containing DNA polymerase, FEN-1endonuclease and a FRET cassette.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleaved flap(e.g., from cleavage of a target-specific probe in a PCR-invasivecleavage assay) with a FRET cassette produces a secondary substrate forthe flap endonuclease, e.g., a FEN-1 enzyme. Once this substrate isformed, the 5′ fluorophore-containing base is cleaved from the cassette,thereby generating a fluorescence signal. In preferred embodiments, aFRET cassette comprises an unpaired 3′ portion to which a cleavageproduct, e.g., a portion of a cleaved flap oligonucleotide, canhybridize to from an invasive cleavage structure cleavable by a FEN-1endonuclease.

A nucleic acid “hairpin” as used herein refers to a region of asingle-stranded nucleic acid that contains a duplex (i.e., base-paired)stem and a loop, formed when the nucleic acid comprises two portionsthat are sufficiently complementary to each other to form a plurality ofconsecutive base pairs.

As used herein, the term “FRET” refers to fluorescence resonance energytransfer, a process in which moieties (e.g., fluorophores) transferenergy e.g., among themselves, or, from a fluorophore to anon-fluorophore (e.g., a quencher molecule). In some circumstances, FRETinvolves an excited donor fluorophore transferring energy to alower-energy acceptor fluorophore via a short-range (e.g., about 10 nmor less) dipole-dipole interaction. In other circumstances, FRETinvolves a loss of fluorescence energy from a donor and an increase influorescence in an acceptor fluorophore. In still other forms of FRET,energy can be exchanged from an excited donor flurophore to anon-fluorescing molecule (e.g., a quenching molecule). FRET is known tothose of skill in the art and has been described (See, e.g., Stryer etal., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol.,246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res573, 103-110, each of which is incorporated herein by reference in itsentirety).

As used herein, the term “FEN-1” in reference to an enzyme refers to anon-polymerase flap endonuclease from a eukaryote or archaeal organism.See, e.g., WO 02/070755, and Kaiser M. W., et al. (1999) J. Biol. Chem.,274:21387, which are incorporated by reference herein in theirentireties for all purposes.

As used herein, the term “FEN-1 activity” refers to any enzymaticactivity of a FEN-1 enzyme, including but not limited to flapendonuclease (FEN), nick exonuclease (EXO), and gap endonuclease (GEN)activities (see, e.g., Shen, et al., BioEssays Volume 27, Issue 7, Pages717-729, incorporated herein by reference).

As used herein, the term “primer annealing” refers to conditions thatpermit oligonucleotide primers to hybridize to template nucleic acidstrands. Conditions for primer annealing vary with the length andsequence of the primer and are generally based upon the T_(m) that isdetermined or calculated for the primer. For example, an annealing stepin an amplification method that involves thermocycling involves reducingthe temperature after a heat denaturation step to a temperature based onthe T_(m) of the primer sequence, for a time sufficient to permit suchannealing.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

The term “real time” as used herein in reference to detection of nucleicacid amplification or signal amplification refers to the detection ormeasurement of the accumulation of products or signal in the reactionwhile the reaction is in progress, e.g., during incubation or thermalcycling. Such detection or measurement may occur continuously, or it mayoccur at a plurality of discrete points during the progress of theamplification reaction, or it may be a combination. For example, in apolymerase chain reaction, detection (e.g., of fluorescence) may occurcontinuously during all or part of thermal cycling, or it may occurtransiently, at one or more points during one or more cycles. In someembodiments, real time detection of PCR is accomplished by determining alevel of fluorescence at the same point (e.g., a time point in thecycle, or temperature step in the cycle) in each of a plurality ofcycles, or in every cycle. Real time detection of amplification may alsobe referred to as detection “during” the amplification reaction.

As used herein, the terms “reverse transcription” and “reversetranscribe” refer to the use of a template-dependent polymerase toproduce a DNA strand complementary to an RNA template.

As used herein, the term “abundance of nucleic acid” refers to theamount of a particular target nucleic acid sequence present in a sampleor aliquot. The amount is generally referred to in terms of mass (e.g.,μg), mass per unit of volume (e.g., μg/μl); copy number (e.g., 1000copies, 1 attomole), or copy number per unit of volume (e.g., 1000copies per ml, 1 attomole per μl). Abundance of a nucleic acid can alsobe expressed as an amount relative to the amount of a standard of knownconcentration or copy number. Measurement of abundance of a nucleic acidmay be on any basis understood by those of skill in the art as being asuitable quantitative representation of nucleic acid abundance,including physical density or the sample, optical density, refractiveproperty, staining properties, or on the basis of the intensity of adetectable label, e.g. a fluorescent label.

The term “amplicon” or “amplified product” refers to a segment ofnucleic acid, generally DNA, generated by an amplification process suchas the PCR process. The terms are also used in reference to RNA segmentsproduced by amplification methods that employ RNA polymerases, such asNASBA, TMA, etc.

The term “amplification plot” as used in reference to a thermal cyclingamplification reaction refers to the plot of signal that is indicativeof amplification, e.g., fluorescence signal, versus cycle number. Whenused in reference to a non-thermal cycling amplification method, anamplification plot generally refers to a plot of the accumulation ofsignal as a function of time.

The term “baseline” as used in reference to an amplification plot refersto the detected signal coming from assembled amplification reactions atprior to incubation or, in the case of PCR, in the initial cycles, inwhich there is little change in signal.

The term “C_(t)” or “threshold cycle” as used herein in reference toreal time detection during an amplification reaction that is thermalcycled refers to the fractional cycle number at which the detectedsignal (e.g., fluorescence) passes the fixed threshold.

The term “no template control” and “no target control” (or “NTC”) asused herein in reference to a control reaction refers to a reaction orsample that does not contain template or target nucleic acid. It is usedto verify amplification quality.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target.”In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.The presence of background template is most often inadvertent. It may bethe result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified away from the sample. Forexample, nucleic acids from organisms other than those to be detectedmay be present as background in a test sample.

A sample “suspected of containing” a nucleic acid may contain or notcontain the target nucleic acid molecule.

As used herein, the term “sample” is used in its broadest sense. Forexample, in some embodiments, it is meant to include a specimen orculture (e.g., microbiological culture), whereas in other embodiments,it is meant to include both biological and environmental samples (e.g.,suspected of comprising a target sequence, gene or template). In someembodiments, a sample may include a specimen of synthetic origin.Samples may be unpurified or may be partially or completely purified orotherwise processed.

The present technology is not limited by the type of biological sampleused or analyzed. The present technology is useful with a variety ofbiological samples including, but are not limited to, tissue (e.g.,organ (e.g., heart, liver, brain, lung, stomach, intestine, spleen,kidney, pancreas, and reproductive organs), glandular, skin, andmuscle), cell (e.g., blood cell (e.g., lymphocyte or erythrocyte),muscle cell, tumor cell, and skin cell), gas, bodily fluid (e.g., bloodor portion thereof, serum, plasma, urine, semen, saliva, etc.), or solid(e.g., stool) samples obtained from a human (e.g., adult, infant, orembryo) or animal (e.g., cattle, poultry, mouse, rat, dog, pig, cat,horse, and the like). In some embodiments, biological samples may besolid food and/or feed products and/or ingredients such as dairy items,vegetables, meat and meat by-products, and waste. Biological samples maybe obtained from all of the various families of domestic animals, aswell as feral or wild animals, including, but not limited to, suchanimals as ungulates, bear, fish, lagomorphs, rodents, etc.

Biological samples also include biopsies and tissue sections (e.g.,biopsy or section of tumor, growth, rash, infection, orparaffin-embedded sections), medical or hospital samples (e.g.,including, but not limited to, blood samples, saliva, buccal swab,cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum,vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine,stool, hair, and sweat), laboratory samples (e.g., subcellularfractions), and forensic samples (e.g., blood or tissue (e.g., spatteror residue), hair and skin cells containing nucleic acids), andarcheological samples (e.g., fossilized organisms, tissue, or cells).

Environmental samples include, but are not limited to, environmentalmaterial such as surface matter, soil, water (e.g., freshwater orseawater), algae, lichens, geological samples, air containing materialscontaining nucleic acids, crystals, and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items.

Samples may be prepared by any desired or suitable method. In someembodiments, nucleic acids are analyzed directly from bodily fluids,stool, or other samples using the methods described in U.S. Pat. Pub.No. 2005/0186588, U.S. patent application Ser. No. 13/470,251, orPCT/US12/37581, each of which is herein incorporated by reference in itsentirety for all purposes.

The above described examples are not, however, to be construed aslimiting the sample (e.g., suspected of comprising a target sequence,gene or template (e.g., the presence or absence of which can bedetermined using the compositions and methods of the presenttechnology)) types applicable to the present technology.

The terms “nucleic acid sequence” and “nucleic acid molecule” as usedherein refer to an oligonucleotide, nucleotide or polynucleotide, andfragments or portions thereof. The terms encompasses sequences thatinclude analogs of DNA and RNA nucleotides, including those listedabove, and also including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,2,6-diaminopurine, and pyrazolo[3,4-d]pyrimidines such as guanineanalogue 6 amino 1H-pyrazolo[3,4d]pyrimidin 4(5H) one (ppG or PPG, alsoSuper G) and the adenine analogue 4 amino 1H-pyrazolo[3,4d]pyrimidine(ppA or PPA). The xanthine analogue 1H-pyrazolo[5,4d]pyrimidin4(5H)-6(7H)-dione (ppX) can also be used. These base analogues, whenpresent in an oligonucleotide, strengthen hybridization and improvemismatch discrimination. All tautomeric forms of naturally-occurringbases, modified bases and base analogues may be included in theoligonucleotide conjugates of the technology. Other modified basesuseful in the present technology include6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, PPPG;6-amino-3-(3-hydroxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one,HOPPPG;6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one,NH2PPPG; 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, PPPA;4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, HOPPPA;4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, NH₂ PPPA;3-prop-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino, (NH₂)₂ PPPA;2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, (NH₂)₂ PPPAOH;3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, (NH₂)₂ PPPANH₂;5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, PU;5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, HOPU;6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, PC;6-amino-5-(3-hydroxyprop-1-yny)-1,3-dihydropyrimidine-2-one, HOPC; and6-amino-5-(3-aminoprop-1-yny)-1,3-dihydropyrimidine-2-one, NH₂PC;5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol,CH₃ OPPPA;6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one,CH₃ OPPPG; 4,(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, Super A;6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one;5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, Super T;3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂PPAI);3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂ PPABr);3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂PPACl);3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPAI);3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPABr); and3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPACl).

A nucleic acid sequence or molecule may be DNA or RNA, of either genomicor synthetic origin, that may be single or double stranded, andrepresent the sense or antisense strand. Thus, nucleic acid sequence maybe dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA made into ssDNA (e.g.,through melting, denaturing, helicases, etc.), A-, B-, or Z-DNA,triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA madeinto ssRNA (e.g., via melting, denaturing, helicases, etc.), messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA,snRNA, microRNA, or protein nucleic acid (PNA).

The present technology is not limited by the type or source of nucleicacid (e.g., sequence or molecule (e.g. target sequence and/oroligonucleotide)) utilized. For example, the nucleic acid sequence maybe amplified or created sequence (e.g., amplification or creation ofnucleic acid sequence via synthesis (e.g., polymerization (e.g., primerextension (e.g., RNA-DNA hybrid primer technology)) and reversetranscription (e.g., of RNA into DNA)) and/or amplification (e.g.,polymerase chain reaction (PCR), rolling circle amplification (RCA),nucleic acid sequence based amplification (NASBA), transcriptionmediated amplification (TMA), ligase chain reaction (LCR), cycling probetechnology, Q-beta replicase, strand displacement amplification (SDA),branched-DNA signal amplification (bDNA), hybrid capture, and helicasedependent amplification).

The terms “nucleotide” and “base” are used interchangeably when used inreference to a nucleic acid sequence, unless indicated otherwise herein.A “nucleobase” is a heterocyclic base such as adenine, guanine,cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or aheterocyclic derivative, analog, or tautomer thereof. A nucleobase canbe naturally occurring or synthetic. Non-limiting examples ofnucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine,hypoxanthine, 8-azapurine, purines substituted at the 8 position withmethyl or bromine, 9-oxo-N6-methyladenine, 2-aminoadenine,7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine,2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine,isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine,5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturallyoccurring nucleobases described in U.S. Pat. Nos. 5,432,272 and6,150,510 and PCT applications WO 92/002258, WO 93/10820, WO 94/22892,and WO 94/24144, and Fasman (“Practical Handbook of Biochemistry andMolecular Biology”, pp. 385-394, 1989, CRC Press, Boca Raton, LO), allherein incorporated by reference in their entireties.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more nucleotides (e.g., deoxyribonucleotides orribonucleotides), preferably at least 5 nucleotides, more preferably atleast about 10-15 nucleotides and more preferably at least about 15 to30 nucleotides, or longer (e.g., oligonucleotides are typically lessthan 200 residues long (e.g., between 15 and 100 nucleotides), however,as used herein, the term is also intended to encompass longerpolynucleotide chains). The exact size will depend on many factors,which in turn depend on the ultimate function or use of theoligonucleotide. Oligonucleotides are often referred to by their length.For example a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes. Oligonucleotides may be generated inany manner, including chemical synthesis, DNA replication, reversetranscription, PCR, or a combination thereof. In some embodiments,oligonucleotides that form invasive cleavage structures are generated ina reaction (e.g., by extension of a primer in an enzymatic extensionreaction).

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction. Asused herein, the terms “complementary” or “complementarity” are used inreference to polynucleotides (e.g., a sequence of two or morenucleotides (e.g., an oligonucleotide or a target nucleic acid)) relatedby the base-pairing rules. For example, the sequence “5′-A-G-T-3′,” iscomplementary to the sequence “3′-T-C-A-5′.” Complementarity may be“partial,” in which only some of the nucleic acid bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acid bases. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon the association of two ormore nucleic acid strands. Either term may also be used in reference toindividual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid sequence (e.g., a targetsequence), in contrast or comparison to the complementarity between therest of the oligonucleotide and the nucleic acid sequence.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Nucleotide analogs, as discussedabove, may be included in the nucleic acids of the present technologyand include. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein, the term “label” refers to any moiety (e.g., chemicalspecies) that can be detected or can lead to a detectable response. Insome preferred embodiments, detection of a label provides quantifiableinformation. Labels can be any known detectable moiety, such as, forexample, a radioactive label (e.g., radionuclides), a ligand (e.g.,biotin or avidin), a chromophore (e.g., a dye or particle that imparts adetectable color), a hapten (e.g., digoxygenin), a mass label, latexbeads, metal particles, a paramagnetic label, a luminescent compound(e.g., bioluminescent, phosphorescent or chemiluminescent labels) or afluorescent compound.

A label may be joined, directly or indirectly, to an oligonucleotide orother biological molecule. Direct labeling can occur through bonds orinteractions that link the label to the oligonucleotide, includingcovalent bonds or non-covalent interactions such as hydrogen bonding,hydrophobic and ionic interactions, or through formation of chelates orcoordination complexes. Indirect labeling can occur through use of abridging moiety or “linker”, such as an antibody or additionaloligonucleotide(s), which is/are either directly or indirectly labeled.

Labels can be used alone or in combination with moieties that cansuppress (e.g., quench), excite, or transfer (e.g., shift) emissionspectra (e.g., fluorescence resonance energy transfer (FRET)) of a label(e.g., a luminescent label).

A “polymerase” is an enzyme generally for joining 3′-OH 5′-triphosphatenucleotides, oligomers, and their analogs. Polymerases include, but arenot limited to, template-dependent DNA-dependent DNA polymerases,DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, andRNA-dependent RNA polymerases. Polymerases include but are not limitedto T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNApolymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1,Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNApolymerase, Vent DNA polymerase (New England Biolabs), Deep Vent DNApolymerase (New England Biolabs), Bst DNA Polymerase Large Fragment,Stoeffel Fragment, 9° N DNA Polymerase, Pfu DNA Polymerase, Tfl DNAPolymerase, RepliPHI Phi29 Polymerase, Tli DNA polymerase, eukaryoticDNA polymerase beta, telomerase, Therminator polymerase (New EnglandBiolabs), KOD HiFi DNA polymerase (Novagen), KOD1 DNA polymerase, Q-betareplicase, terminal transferase, AMV reverse transcriptase, M-MLVreverse transcriptase, Phi6 reverse transcriptase, HIV-1 reversetranscriptase, novel polymerases discovered by bioprospecting, andpolymerases cited in US 2007/0048748, U.S. Pat. Nos. 6,329,178;6,602,695; and 6,395,524 (incorporated by reference). These polymerasesinclude wild-type, mutant isoforms, and genetically engineered variants.

A “DNA polymerase” is a polymerase that produces DNA fromdeoxynucleotide monomers (dNTPs). “Eubacterial DNA polymerase” as usedherein refers to the Pol A type DNA polymerases (repair polymerases)from Eubacteria, including but not limited to DNA Polymerase I from E.coli, Taq DNA polymerase from Thermus aquaticus and DNA Pol I enzymesfrom other members of genus Thermus, and other eubacterial species etc.

As used herein, the term “target” refers to a nucleic acid species ornucleic acid sequence or structure to be detected or characterized.

Accordingly, as used herein, “non-target”, e.g., as it is used todescribe a nucleic acid such as a DNA, refers to nucleic acid that maybe present in a reaction, that is not the subject of detection orcharacterization by the reaction. In some embodiments, non-targetnucleic acid may refer to nucleic acid present in a sample that doesnot, e.g., contain a target sequence, while in some embodiments,non-target may refer to exogenous nucleic acid, i.e., nucleic acid thatdoes not originate from a sample containing or suspected of containing atarget nucleic acid, and that is added to a reaction, e.g., to normalizethe activity of an enzyme (e.g., polymerase) to reduce variability inthe performance of the enzyme in the reaction.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel.

As used herein, the term “flap assay reagents” or “invasive cleavageassay reagents” refers to all reagents that are required for performinga flap assay or invasive cleavage assay on a substrate. As is known inthe art, flap assays generally include an invasive oligonucleotide, aflap oligonucleotide, a flap endonuclease and, optionally, a FRETcassette, as described above. Flap assay reagents may optionally containa target to which the invasive oligonucleotide and flap oligonucleotidebind.

As used herein, the term “flap oligonucleotide” refers to anoligonucleotide cleavable in a detection assay, such as an invasivecleavage assay, by a flap endonuclease. In preferred embodiments, a flapoligonucleotide forms an invasive cleavage structure with other nucleicacids, e.g., a target nucleic acid and an invasive oligonucleotide.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is technology relating to the amplification-baseddetection of nucleic acids and particularly, but not exclusively, tomethods and compositions for altering the behavior of calibrationstandards to mimic natural samples and minimize variability in theactivity of manufactured polymerases, such as Taq polymerase, e.g., whenusing combined amplification and invasive cleavage assay reactions.

Typically, when a small change in a reagent produces a shift in astandard curve (e.g., a change in the slope and/or intercept), the shiftmay indicate a suboptimal condition relative to one or more componentsof the reaction. As such, the conventional practice is to optimize theassay components and their concentrations.

In the case of standard curves changing in response to changes in asource, manufacturer, or manufacturer's lot of Taq polymerase, it wasinitially suspected that slight lot-to-lot variations in the Taqconcentration resulted in the detected differences in the standardcurves. To test this hypothesis, the K-ras (KRAS) gene and a gene panelcalled “ANB,” consisting of ACTB (β-actin, which typically serves as areference standard in the assays), NDRG4 (member of the N-mycdownregulated gene family), and BMP3 (bone morphogenetic protein 3),were quantified in a QuARTS reaction using Taq DNA polymerase from threedifferent lots and at concentrations of 0.04, 0.06, 0.08, and 1 unit/μl.Surprisingly, the data collected (Table 1) show that adjusting the Taqconcentration did not correct errors in quantifying the ANB and KRASstrands.

TABLE 1 Mean strands FAM HEX Quasar 670 Taq lot Taq lot Taq lot AssaySampleID Taq U/ul 11256007 31614211 32171002 11256007 31614211 3217100211256007 31614211 32171002 ANB 200 strands 0.04 3,973 3,217 2,767 5,3993,951 3,740 12,026 10,884 4,334 per 0.06 4,723 3,631 3,017 6,462 4,5484,443 11,046 9,979 5,062 reaction 0.08 3,189 4,314 3,989 6,253 5,4285,278 5,602 9,126 5,558 spiked 0.1 3,323 4,504 4,842 5,690 5,201 6,7135,056 8,512 7,330 Kras sDNA 0.04 32 9 13 19 0 19 7,041 3,729 7,144 0.069 9 6 3 9 7 12,895 5,917 11,801 0.08 4 25 0 1 16 0 12,506 8,122 14,9370.1 4 15 0 1 8 0 10,519 9,949 9,101 200 strands 0.04 541 371 525 1,056722 1,335 7,499 4,350 7,678 per 0.06 789 441 679 1,746 1,232 1,56412,173 7,060 14,226 reaction 0.08 578 1,046 218 2,705 2,051 1,300 12,31510,831 17,599 spiked 0.1 358 1,166 88 2,322 1,927 434 12,660 13,72210,327

In the table above, for ANB assays (see left column), the FAM signalindicates the NDRG4 target, HEX indicates the BMP3 target, and Quasar670 indicates the ACTB target; for the Kras assays, the FAM signalindicates KRAS 35T, 34T, 38 targets, HEX indicates KRAS 35A, 35C, 34A34C targets, and Quasar 670 indicates ACTB targets. These data areaveraged signals from duplicate reactions for normal stool DNA (“sDNA”)samples, and triplicate reactions for the samples in which plasmid DNAwas spiked into sDNA. The amount of normal stool DNA used is 10 μl of apreparation such as would typically be produced by DNA capture from astool sample for testing, e.g., using a QuARTS assay.

The data in Table 1 shows that there is notable variation in the signalgenerated by the use of the same unit amounts of different lots of theTaq DNA polymerase. Each row in Table 1 is associated with a particularnumber of units of Taq DNA polymerase per reaction, but using differentlots of Taq shows that different lots produced highly variable signalsfrom the same input target DNA. For example, when 0.04 u/μl was used inthe ANB assay, the signal in the Quasar 670 channel showed more thantwice as much signal from lots 11256007 and 316142211 as from lot32171002.

If the polymerases from different lots exhibited the same performancebut varied only in concentration, one would expect that in the spikedsamples, one would calculate out the same figures for the quantity ofinput DNA (number of strands) regardless of the lot of enzyme used, evenif the gross fluorescence signals varied, and that the differencesbetween lots could be addressed by adjusting the amount of enzyme addedwhen switching between lots. Table 1 shows, however, that thedifferences in performance cannot be compensated for by adjusting enzymeconcentration. Using higher or lower amounts of enzyme from one lot didnot result in reaction performance matching the performance level of adifferent lot of the same enzyme. For example, the performance of 0.06units of lot 11256007 is not the same as, or close to, the performanceof 0.04, 0.06, 0.08, or 0.1 units of the other lots of Taq tested,demonstrating that the variable performance cannot be attributed todifferences in the unit concentrations of the Taq DNA polymerase.

Accordingly, other solutions to this problem in variability are providedin the description of the present technology. In the followingdescription, the section headings used herein are for organizationalpurposes only and are not to be construed as limiting the describedsubject matter in any way.

Embodiments of the Technology

1. Methods to Minimize DNA Polymerase Variability

Provided herein is technology related to minimizing polymerasevariability by adding extraneous, non-target DNA to samples. While thetechnology is described herein in particular aspects as it relates toPCR-invasive cleavage assays, such as QuARTS assay technology, thetechnology is not limited in its application to PCR-invasive cleavageassays and QuARTS assays, but finds use in any technology in which apolymerase is employed. Indeed, it is contemplated that the technologyfinds use in any test, assay, or other technology in which an enzymecontacts a nucleic acid, and, in particular, in any test, assay, orother technology in which an enzyme contacts a nucleic acid according toa degree of sequence specificity to differentiate target frombackground. Examples of such technologies are ligases, restrictionenzymes, DNA polymerases, RNA polymerases, reverse transcriptases,telomerases, recombinases, DNA and/or RNA editing enzymes, RNA splicingenzymes, methylases, enzymes for poly-A tail addition, nucleases, miRNAand siRNA processing enzymes, and the like.

While not limiting the invention to any particular mechanism of action,it has been observed that the presence of hairpin oligonucleotides(e.g., hairpin FRET cassettes as used, for example, in some embodimentsof invasive cleavage detection assays) may have an inhibiting effect onDNA polymerase present in the same vessel, as assessed by sample andsignal amplification. See, e.g., U.S. Patent Publication 2006/0147955 toAllawi, which is incorporated herein by reference for all purposes.Allawi et al. observed that when PCR and invasive cleavage assaycomponents were combined, the hairpin FRET oligonucleotides affectedpolymerase performance. As such, the present technology findsapplication in DNA polymerase-based assays conducted in the presence ofhairpin-containing DNAs, including but not limited to PCR-invasivecleavage assays, such as QuARTS assays.

According to the technology, purified exogenous non-target DNA is addedto samples before and/or while contacting the samples with an enzymesuch as a polymerase. The non-target DNA is added to the sample orreaction mixture, for example, at a concentration of approximately 2 to20 ng per μl of reaction mixture, preferably approximately 6 toapproximately 7 ng per μl of reaction mixture, when approximately 0.01to 1.0 U/μl of enzyme, e.g., 0.05 U/μl of enzyme (e.g., a polymerasesuch as, e.g., Taq polymerase) is used in the assay. It is contemplatedthat a amounts of from 10 ng to 1 μg of the purified exogenousnon-target DNA are added depending on the particular assay system, forexample, when considering the particular enzyme and enzymeconcentrations, concentrations of other assay components (e.g., primers,salts, target, etc.) present, and relevant physical and chemical factorssuch as temperature, pressure, water activity, volume, presence ofinhibitors, etc.

2. Compositions for Minimizing Polymerase Variability

In another aspect, compositions according to the technology comprisenon-target DNA, and, in some embodiments, one or more of a buffer, oneor more salts, a preservative (e.g., sodium azide), a chelator (EDTA,EGTA, BAPTA, etc.), a free radical scavenger, etc., typically in anaqueous solution. However, non-aqueous and/or organic solutions (e.g.,dimethyl sulfate) are also contemplated as are dried compositionsprovided, e.g., as a powder (e.g., freeze-dried, lyophilized, etc.). Thetechnology includes embodiments of compositions comprising thenon-target DNA as a composition to add to an assay sample (e.g., acomposition comprising non-target DNA before it has been added to asample) and embodiments of compositions comprising the non-target DNAand the components of the assay sample (e.g., after addition of thenon-target DNA to the sample).

It is contemplated that the technology encompasses the use of DNA fromany source. Specific examples of the technology provided herein relateto the use of fish (e.g., herring) DNA and/or mammalian (e.g., mouse)DNA. However, in some embodiments, the non-target nucleic acid isisolated from another source, e.g., a natural source (e.g., an animal, abacterium, an archaeon, a virus, a eukaryote (e.g., a yeast), a plant, apool or mixture of one or more of these, etc., as taken from anenvironmental or biological sample, from a culture, from a recombinantsource, or a mixture of one or more of these) or, in some embodiments,the nucleic acid is synthetic (e.g., produced chemically (e.g., by amachine) and optionally comprises one or more sequences present in anatural source and/or random sequences). In some embodiments, thesequence of the non-target nucleic acid is known and in some embodimentsthe sequence of the non-target nucleic acid is not known or is partiallyknown. In some embodiments, the non-target nucleic acid is labeled ortagged such that it can be distinguished or isolated from target nucleicacid or from assay sample nucleic acid that is not the added non-targetnucleic acid. In some embodiments, the non-target nucleic acid ismodified such that it is not a substrate for the assay to which thenon-target nucleic acid is added. For example, non-target DNA with ablocked 3′ end is not a substrate for primer extension by a polymerase.In addition, in some embodiments, certain modifications of the bases ofa non-target nucleic acid inhibit the binding of a primer, thusinhibiting the non-target nucleic acid from acting as a template. Insome embodiments, the non-target nucleic acid is susceptible todigestion and/or transformation to a state that does not interfere withthe downstream processing of a sample.

The extent of purification of the non-target nucleic acid varies. Insome embodiments, the non-target nucleic acid is DNA substantially oressentially free of RNA and in some embodiments the non-target nucleicacid is RNA substantially free or essentially free of DNA. In someembodiments the non-target nucleic acid is substantially or essentiallyfree of proteins. In specific embodiments, the nucleic acid issubstantially free of enzymes that degrade a nucleic acid and/or thatinhibit and/or deleteriously affect an assay of a sample to which thenon-target nucleic acid is added. Similarly, some embodiments includecompositions are essentially free of a component such as, e.g., an ion,a salt, a lipid, or some other component that results in the degradationor sequestration of DNA and/or that inhibits an assay for the detectionof the target nucleic acid.

In some embodiments, compositions comprise an aqueous buffer medium thatis optimized for the particular non-target DNA and/or polymerase in theassay in which the non-target DNA is to be used. Buffer systems includecitrate buffers, acetate buffers, borate buffers, and phosphate buffers.Other exemplary buffers include citric acid, sodium citrate, sodiumacetate, acetic acid, sodium phosphate and phosphoric acid, sodiumascorbate, tartartic acid, maleic acid, glycine, sodium lactate, lacticacid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid,sodium succinate and succinic acid, histidine, and sodium benzoate andbenzoic acid. Preferred exemplary buffering agents that are present inembodiments of compositions according to the technology describedinclude Tris, Tricine, HEPES, MOPS, and the like, where the amount ofbuffering agent will typically range from about 5 to 150 mM, usuallyfrom about 10 to 100 mM, and more usually from about 20 to 50 mM, wherein certain preferred embodiments the buffering agent will be present inan amount sufficient to provide a pH ranging from about 6.0 to 9.5,e.g., a pH of approximately 8.0.

In some embodiments, the compositions include a source of monovalentions and/or a source of divalent cations. Any convenient source ofmonovalent ions, such as potassium chloride, sodium chloride, potassiumacetate, sodium acetate, potassium glutamate, ammonium chloride,ammonium sulfate, and the like may be employed. Divalent ions are, e.g.,magnesium, manganese, calcium, etc.

In some embodiments, the compositions comprise one or more chelatingagents, such as ethylenediaminetetraacetic acid (also synonymous withEDTA, edetic acid, versene acid, and sequestrene), and EDTA derivatives,such as dipotassium edetate, disodium edetate, edetate calcium disodium,sodium edetate, trisodium edetate, and potassium edetate. Otherchelating agents include EGTA, BAPTA, citric acid and derivativesthereof. Citric acid also is known as citric acid monohydrate.Derivatives of citric acid include anhydrous citric acid andtrisodiumcitrate-dihydrate. Still other chelating agents includeniacinamide and derivatives thereof, sodium desoxycholate andderivatives thereof.

In some embodiments, the compositions comprise an antioxidant.Antioxidants are well known to those of ordinary skill in the art andinclude materials such as ascorbic acid, ascorbic acid derivatives(e.g., ascorbylpalmitate, ascorbylstearate, sodium ascorbate, calciumascorbate, etc.), butylated hydroxy anisole, buylated hydroxy toluene,alkylgallate, sodium meta-bisulfate, sodium bisulfate, sodiumdithionite, sodium thioglycollic acid, sodium formaldehyde sulfoxylate,tocopherol and derivatives thereof, (d-alpha tocopherol, d-alphatocopherol acetate, dl-alpha tocopherol acetate, d-alpha tocopherolsuccinate, beta tocopherol, delta tocopherol, gamma tocopherol, andd-alpha tocopherol polyoxyethylene glycol 1000 succinate)monothioglycerol, and sodium sulfite. Such materials are typically addedin ranges from 0.01 to 2.0%.

In some embodiments, the compositions comprise a cryoprotectant. Commoncryoprotecting agents include histidine, polyethylene glycol, polyvinylpyrrolidine, lactose, sucrose, mannitol, and polyols. Accordingly, insome embodiments, compositions are stored frozen.

In some embodiments, the compositions comprise a preservative. Commonpreservatives include chlorobutanol, parabens, thimerosol, benzylalcohol, and phenol. Suitable preservatives include but are not limitedto: chlorobutanol (0.3-0.9% w/v), parabens (0.01-5.0%), thimerosal(0.004-0.2%), benzyl alcohol (0.5-5%), phenol (0.1-1.0%), and the like.In some embodiments, compositions comprise sodium azide (NaN₃), e.g., ata concentration of approximately 0.025%.

Compositions are prepared in some embodiments as concentrated solutions,e.g., as a 2×, 5×, 10×, 20×, 25×, 50×, 100×, or more concentrated,solution. As an example, a 2× solution is added to an equal part ofanother solution to provide the non-target DNA at the finalconcentration at which it is used (e.g., at 1×). For a workingconcentration of 200 ng of non-target DNA per 20 μl reaction sample, a2× concentrated solution is prepared to comprise 400 ng/20 μl, or 20ng/μl non-target DNA. For this same reaction volume, a 10× solution isprepared at 2000 ng per 20 μl, or at 100 ng/μl, etc. Some embodimentsprovide that compositions are protected from light and/or ionizingradiation.

3. Uses

In some embodiments, the methods and compositions provided herein finduse in an assay for detecting and/or quantifying a nucleic acid. Forexample, the technology finds use in assays based, for example, on PCR,INVADER assay, PCR-invasive cleavage assays and/or QuARTS assay methods.In particular, the technology finds use in quantitative assays in whichstandards are used to construct a standard curve.

3.1. Use in QuARTS Assay

In some embodiments, the technology finds use to normalize enzyme (e.g.,polymerase) activity to reduce enzyme-associated variability in a QuARTSassay. The QuARTS assay is often used to quantify the number of copiesof a particular DNA in a sample. The QuARTS technology combines apolymerase-based target DNA amplification process with an invasivecleavage-based signal amplification process. Fluorescence signalgenerated by the QuARTS reaction is monitored in a fashion similar toreal-time PCR. During each amplification cycle, three sequentialchemical reactions occur in each assay well, with the first and secondreactions occurring on target DNA templates and the third occurring on asynthetic DNA target labeled with a fluorophore and quencher dyes, thusforming a fluorescence resonance energy transfer (FRET) donor andacceptor pair. The first reaction produces amplified target with apolymerase and oligonucleotide primers, and the second reaction uses ahighly structure-specific 5′-flap endonuclease-1 (FEN-1) enzyme reactionto release a 5′-flap sequence from a target-specific oligonucleotideprobe that binds to the product of the polymerase reaction, forming anoverlap flap substrate. In the third reaction, the cleaved flap binds toa specially designed oligonucleotide containing a fluorophore andquencher closely linked in a FRET pair such that the fluorescence isquenched (FRET cassette). The released probe flap hybridizes in a mannerthat forms an overlap flap substrate that allows the FEN-1 enzyme tocleave the 5′-flap containing the fluorophore, thus releasing it fromthe quencher molecule. The released fluorophore generates fluorescencesignal to be detected. During the second and third reactions, the FEN-1reaction can cut multiple probes per target, generating multiple5′-flaps and multiple FRET cassettes per cleaved 5′-flap, giving rise toadditional linear signal amplification to the overall reaction.

In some applications, the assay is designed for methylated DNA analysis.In some configurations, each assay is designed to detect multiple genes,e.g., 3 genes reporting to 3 distinct fluorescent dyes. See, e.g., Zou,et al., (2012) “Quantification of Methylated Markers with a MultiplexMethylation-Specific Technology”, Clinical Chemistry 58: 2, incorporatedherein by reference for all purposes.

An exemplary QuARTS reaction typically comprises approximately 400-600nmol/l (e.g., 500 nmol/l) of each primer and detection probe,approximately 100 nmol/l of the invasive oligonucleotide, approximately600-700 nmol/l of each FAM (e.g., as supplied commercially by Hologic),HEX (e.g., as supplied commercially by BioSearch Technologies, IDT), andQuasar 670 (e.g., as supplied commercially by BioSearch Technologies)FRET cassettes, 6.675 ng/μl FEN-1 (e.g., Cleavase® (e.g., 2.0), Hologic,Inc.), 1 unit Taq DNA polymerase in a 30 μl reaction volume (e.g.,GoTaq® DNA polymerase, Promega Corp., Madison, Wis.), 10 mmol/l3-(n-morpholino)propanesulfonic acid (MOPS), 7.5 mmol/l MgCl₂, and 250μmol/l of each dNTP. Exemplary QuARTS cycling conditions consist of aninitial incubation at 95° C. for 3 minutes, followed by 10 cycles of 95°C. for 20 seconds, 67° C. for 30 seconds, and 70° C. for 30 seconds.After completion of the 10 cycles, an additional 37 cycles at 95° C. for20 seconds, 53° C. for 1 minute, 70° C. for 30 seconds, and 40° C. for30 seconds are typically performed. In some applications, analysis ofthe quantification cycle (C_(q)) provides a measure of the initialnumber of target DNA strands (e.g., copy number) in the sample.

The technology of the present invention finds use in normalizing theactivity and reducing variability in the performance of polymerases,e.g., that are used in a QuARTS assay, for example, by use of themethods and compositions described herein. These embodiments are furtherunderstood by the illustrative examples provided below.

EXPERIMENTAL EXAMPLES Example 1

During the development of embodiments of the technology provided herein,it was hypothesized that the addition of exogenous non-target containingDNA would minimize manufacturing variability in Taq polymerase and/orresemble the non-target DNA that is present in some complex samples suchas stool DNA (sDNA) samples that are often used for colorectal cancerscreening. To test this hypothesis, varying amounts of mouse and herringDNA were added to KRAS calibrators at 10, 100, 1000 and 10,000 copiesper reaction (n=4 replicates each). Then, using two different lots ofTaq polymerase (designated “DEV” and “New Pro”), the differences betweenthe standard curves' slopes and intercepts were examined. Concentrationsof the reaction components (in nM, except as indicated for dNTPs) areshown below:

KRAS Reverse primer 300 KRAS 35C probe 500 ACTB Reverse primer 500 KRAS34A probe 500 KRAS 34A forward primer 250 KRAS 34C probe 500 KRAS 34Tforward primer 250 KRAS 35T probe 500 KRAS 34C forward primer 250 KRAS34T probe 500 KRAS 35A forward primer 250 KRAS 38A probe 250 KRAS 35Tforward primer 250 ACTB probe 500 KRAS 35C forward primer 250 A5 HEXFRET 100 KRAS 38A forward primer 250 A7 FAM FRET 100 ACTB WT forwardprimer 500 A1 Quasar670 FRET 150 KRAS 35A probe 500 25 mM dNTPs 250 (uM)Reaction volume = 30 μl,Dye Reporting:

-   -   KRAS 35A, 35C, 34A 34C=HEX    -   KRAS 35T, 34T, 38A=FAM    -   ACTB=Quasar        The cycling parameters for these QuARTS reactions are follows:

Stage Temp (° C.) Time (minutes) Ramp Rate Number of Pre-Incubation 953:00 100% 1 Amplification 1 95 0:20 100% 10 63 0:30 100% 70 0:30 100%Amplification 2 95 0:20 100% 35 53 1:00 100% 70 0:30 100% Cooling 400:30 100% 1

Results are shown in FIGS. 2A and 2B. These data show that addition ofherring DNA decreases the differences between the slopes (FIG. 2A) andintercepts (FIG. 2B) of the signal from the calibrators, and that as theamount of herring DNA is increased from 1 to 4 ng/reaction, thedifferences between the slopes and intercepts decrease. Mouse DNA hadthe same effect (data not shown).

Example 2

tRNA is often added to calibrator samples used in quantitative PCR,e.g., to minimize sticking of calibrator DNA to vessels. To test whethertRNA had the same effect on polymerase performance in a PCR-invasivecleavage assay as observed with herring DNA, QuARTS reactions wereconducted as described above, but in the presence of 20 ng/μl of tRNAinstead of 4 ng/μl of herring DNA. The results are shown in FIG. 3.Calibration plots for the two lots of Taq are nearly identical in thepresence of herring DNA (FIG. 3B), but not in the presence of tRNA (FIG.3A), indicating that the performance variation is not due to binding ofthe calibrator DNA to the vessels.

Example 3

During the development of embodiments of the technology, data werecollected from experiments in which standard curves were generated fromdifferent lots of Taq in the presence of USB/Affymetrix fish (salmon)DNA (Cleveland, Ohio). A comparison of two lots of Promega “Hot Start GoTaq” (indicated as “DEV” and “Pnew”, and present at concentrations of0.03, 0.05, and 0.07 U/μl; Promega Corp., Madison, Wis.) in the KRASQuARTS assay with the addition of 200 ng/reaction of USB/Affymetrix fishDNA showed that the standard curves were nearly identical between thetwo Taq lots for all three dyes (Table 3). FIG. 4 shows plots of thedata from the 0.05 and 0.07 U/μl reaction sets.

TABLE 3 delta Cp, DEV lot Taq − Pr new lot Taq Taq concentration 0.03U/μL Taq concentration 0.05 U/μL Taq concentration 0.07 U/μL Target FAMHEX Quasar 670 FAM HEX Quasar 670 FAM HEX Quasar 670 KRAS 1e1 cp/rxn−0.58 −0.05 −0.26 0.17 −0.17 0.11 0.01 −0.38 −0.28 Calibrator: 1e2cp/rxn −0.01 −0.11 −0.05 0.36 0.32 −0.13 0.75 1.22 0.41 38A, 35C, 1e3cp/rxn −0.20 −0.29 −0.20 −0.06 0.02 −0.06 0.94 1.2 0.78 ACTB WT 1e4cp/rxn −0.17 −0.26 −0.17 −0.30 −0.18 −0.28 0.57 0.68 0.45 1e5 cp/rxn−0.13 −0.22 −0.26 −0.26 −0.20 −0.28 −0.16 0.03 −0.14 Controls KRAS WT1e5 0.13 0.00 0.00 0.62 0.51 0.00 0.06 0.43 0 NTC NA NA NA NA NA NA NANA NA Average difference in Cp −0.16 −0.15 −0.16 0.09 0.05 −0.11 0.360.53 0.20 values, DEV − Pr new Taq lots

A comparison of Roche fish (cod and herring sperm) DNA (Roche AppliedScience, Mannheim, Germany) and USB/Affymetrix fish (salmon) DNA at 200ng/reaction with 0.05 U/μl of Taq DNA polymerase result in nearlyidentical signals for KRAS samples across the two different Taq lots(Table 4).

TABLE 4 Fish DNA Strands FAM Strands HEX Strands Quasar Strands FAMStrands HEX Strands Quasar Sample Name Supplier Taq Lot Mean Mean MeanCV CV CV N sDNA Roche DEV 7.94 5.76 10238.66 15.88 40.85 9.31 4.00 PrNew 8.90 6.04 9269.49 7.03 83.68 7.89 2.00 USB DEV 8.03 6.29 9169.3232.00 40.99 7.64 4.00 Pr New 10.68 8.21 9506.81 22.78 58.11 5.08 2.00sDNA + 1e2 Roche DEV 551.87 540.18 10337.09 2.27 2.76 10.66 2.00 Pr New448.16 475.11 10013.05 19.87 19.28 15.70 4.00 USB DEV 491.86 535.899250.39 19.48 5.49 4.53 2.00 Pr New 469.66 571.85 10210.97 21.11 14.4115.08 4.00

Example 4

During the development of embodiments of the technology, tests wereconducted to assess if including fish DNA in samples affects thedetection of targets in the assay. In particular, a QuARTS reaction toquantify the ANB panel was performed using 0.05 and 0.07 U/μl Taqpolymerase in the presence of 200 ng Roche fish DNA per reaction. Thetests quantified the ANB panel in stool DNA and stool DNA spiked with100 copies of ANB borne on a plasmid. Results show no change in thecalibrators or sample recoveries (Table 5). NTC=no-template negativecontrol.

TABLE 5 Mean CV Sample Name Taq Lot Taq Conc fish DNA Strands FAMStrands HEX Strands Quasar Strands FAM Strands HEX Strands Quasar N NTCDev 0.05 0 0 0 0 Missing Missing Missing 8 200 0 0 0 Missing MissingMissing 8 0.07 0 0 0 0 Missing Missing Missing 8 200 0 0 0 MissingMissing Missing 8 Pr New 0.05 200 0 0 0 Missing Missing Missing 8 sDNADev 0.05 0 26 13 2020 28.04 3.22 3.99 2 200 19 5 1785 12.10 41.70 6.60 20.07 0 39 16 2100 1.01 44.62 0.16 2 200 36 36 2023 21.11 5.15 0.57 2 PrNew 0.05 200 19 20 1964 61.37 44.10 4.75 2 sDNA + 1e2 Dev 0.05 0 326 3352120 13.76 18.95 3.05 2 200 323 329 2119 5.27 8.46 3.88 2 0.07 0 399 3642491 0.35 4.03 6.19 2 200 343 387 2176 15.33 11.98 15.31 2 Pr New 0.05200 400 407 2277 3.99 8.77 3.62 2

The results indicate that including fish DNA in the dilution buffer ofstandards or calibrator samples reduces the variations in standardcurves that result from variability in the performance of differentproduction lots of Taq DNA polymerase. Additional experiments showedthat this result was not achievable by adding tRNA alone, by using Taqfrom different vendors (data not shown), or by titrating the amount ofTaq per reaction.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entireties for all purposes.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which the various embodiments described herein belongs.When definitions of terms in incorporated references appear to differfrom the definitions provided in the present teachings, the definitionprovided in the present teachings shall control.

Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that thetechnology as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the technology that are obvious to those skilled inpharmacology, biochemistry, medical science, or related fields areintended to be within the scope of the following claims.

We claim:
 1. A reaction mixture comprising: a) an amount of humangenomic target nucleic acid; b) purified exogenous non-target DNAisolated from fish at a concentration of approximately 2 to 20 nanogramsper pi of reaction mixture; and c) PCR amplification assay reagentscomprising: i) thermostable DNA polymerase; ii) dNTPs; iii) a firstprimer and a second primer configured for amplifying a DNA product fromsaid target nucleic acid; iv) an oligonucleotide with a fluorescentlabel; and v) a flap endonuclease, wherein said reaction mixture ischaracterized in that it can amplify said DNA product from said targetnucleic acid and produce an amount of amplified DNA product proportionalto the amount of said target nucleic acid in said reaction mixture. 2.The reaction mixture of claim 1, wherein said purified exogenousnon-target DNA isolated from fish comprises DNA isolated from herringand/or cod and/or salmon.
 3. The reaction mixture of claim 1, whereinsaid reaction mixture comprises purified exogenous non-target DNAisolated from fish at a concentration of approximately 6 to 7 nanogramsper μl of reaction mixture.
 4. The reaction mixture of claim 1, whereinsaid thermostable DNA polymerase is a eubacterial DNA polymerase.
 5. Thereaction mixture of claim 4, wherein said thermostable DNA polymerase isfrom Thermus aquaticus.
 6. The reaction mixture of claim 1, wherein theDNA polymerase is modified for hot start PCR.
 7. The reaction mixture ofclaim 1, wherein said flap endonuclease is a FEN-1 endonuclease.